Registration Dossier

Administrative data

Description of key information

In an Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422) with trichloro(N,N-dimethyloctylamine)boron (TK 12146) the NOAEL for systemic toxicity was found to be 1000 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 09 July 2014 and 10 March 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
See principles of method if other than Guideline part.
Principles of method if other than guideline:
Deviation No.1
Animal Husbandry
Environment
The Study Plan target value for relative humidity controls was 50 ± 20%. The target range for relative humidity was exceeded on eighteen separate days during the course of the study with the maximum humidity achieved being 79.69% RH. While it is accepted that these deviations from the target range for relative humidity were less than ideal, overall it was considered that these^ deviations had no adverse impact on the scientific purpose of the study.

Deviation No 2
Functional Test
Eight animals from the third lactation set of females were assessed using activity monitors for 30 minutes over which five data acquisitions were recorded for each animal. The acquired data was printed out, signed and dated, it was also saved in excel format at the end of the assessment. When the printed data was checked at a later date it was discovered that some of the data was missing from the printout, only twenty-six data acquisition points were present
rather than the forty that was expected.
The missing data was recovered by transferring the saved file to a PC at which point it was printed signed and dated. The retention of the data and file transfer has not been validated so this is considered to be a GLP exception. This data has been reported although no claim of GLP compliance will be made for this part of the work. This deviation was considered not to affect the scientific integrity of the study.

Deviation No 3
Clinical Observations
On Day 43 of the study the post dose observations were not performed on the required last six animals in each dose group due to a technician error. No adverse clinical signs were noted on this study therefore the omission was considered not to affect the scientific integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Animals and Animal Husbandry
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for six days during which time their health status was assessed. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 299 to 357g, the females weighed 203 to 230g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
Test Item Preparation
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.

Samples of the test item formulations were taken and analyzed for concentration of trichloro(N,N-dimethyloctylamine)boron (TK 12146) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Introduction
The test item concentration in the test samples was determined by gas chromatography using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Test item
The test item described in the main part of the study was also used as the analytical standard.

Analytical procedure
Preparation of standard solutions
Stock solutions of test item in acetonitrile were prepared for external calibration. An aliquot, 100 mg of test item, was accurately weighed into a 100 mL volumetric flask and brought to volume with acetonitrile to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in acetonitrile with a concentration of 0.1 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The formulations received were extracted with acetonitrile. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with acetonitrile this was then ultra-sonicated for 15 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with acetonitrile to achieve the working concentration.

Preparation of accuracy samples
Samples of arachis oil BP wereaccurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as the test samples.

Preparation of linearity standards
A range of standard dose solutions were prepared in acetonitrile from a stock solution of 1.017 mg/mL by serial dilution covering the concentration range of 0 to 0.2034 mg/mL.

Instrumental set up
GC system: Agilent Technologies 5890, incorporating autosampler and workstation
Colum: ZB-5 (30 m x 0.53 m id x 5 µm film)
Oven temperature program: 50 °C for 0 minutes with 10 °C / minute to 300 °C for 5 minutes.
Injection temperature: 250 °C
Flame ionisation detector temperature: 250 °C
Injection volume: 1 µ L
Retention time: ~ 10 mins

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.

Results
Validation of analytical method
Specificity
The control dose samples and an analysed solvent blank showed not significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration; hence the specificity of the method by retention time was confirmed.

Linearity
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. The data was found to have a linear correlation within the calibration range. The R2 fit of the calibration curve to the data was 0.999 and considered to be acceptable.

Accuracy
The fortified samples of arachis oil BP were found to have a recovery value of ± 10 % of the fortification.

Test item formulations
The formulations investigated during the study were found to comprise test item in the range of 101 % to 110 % and thus the required content limit of ± 10 % with reference to the nominal content was met.
The test item was found to be stable in the formulations when kept for 14 days at room temperature due to results which met the variation limit of 10 % from the time-zero mean.
In conclusion, the results indicated the accurate use of the test item and arachis oil BP as vehicle during the study. The formulations were found to be homogenously prepared and sufficient formulation stability under storage conditions was proven.

Discussion
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven to be suitable for use.
Duration of treatment / exposure:
Up to seven weeks
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Twelve/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen based on the results of previous toxicity work (Harlan Laboratories Ltd., Project Number 41401348). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43 or Day 44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Positive control:
None
Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.


Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli


Behavioral Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:

Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.


Functional Performance Tests
Motor Activity.
Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength.
An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).

The following parameters were observed:
Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.


Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.


Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.


Water Consumption
Water intake was measured daily for males from Day 20 onwards and during gestation and lactation for females.


Reproductive Performance
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.


Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:

i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:

i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)


Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Laboratory Investigations
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

Hematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:

Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)

Total leukocyte count (WBC)

Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)

Reticulocyte count (Retic)
- Methylene blue stained slides were prepared but reticulocytes were not assessed


Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:

Urea, Calcium (Ca++), Glucose, Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili), Bile acids.
Sacrifice and pathology:
Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or Day 44. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced for non-pregnant females, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group:

Adrenals Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation).

Tissues shown below were weighed from all remaining animals:
Prostate
Seminal vesicles
Epididymides
Testes
Ovaries
Uterus (weighed with Cervix)
Pituitary (post fixation)

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated.
Adrenals, Muscle (skeletal), Aorta (thoracic), Ovaries, Bone & bone marrow (femur including stifle joint), Pancreas, Bone & bone marrow (sternum), Pituitary, Brain (including cerebrum, cerebellum and pons), Prostate, Cecum, Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides•, Skin (hind limb), Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes*, Gross lesions, Spleen, Heart, Stomach, Ileum (including peyer’s patches), Thyroid/parathyroid, Jejunum, Trachea, Kidneys, Testes•, Liver, Thymus, Lungs (with bronchi) #, Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus/Cervix, Mammary gland , Vagina.
• = preserved in Modified Davidsons fluid
* = eyes fixed in Davidson’s fluid

Tissues shown below were preserved from all remaining animals:
Ovaries
Pituitary
Prostate
Coagulating gland
Seminal vesicles
Epididymides
Gross lesions
Testes
Uterus/Cervix
Vagina
Mammary gland

Tissues were dispatched to the Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). The tissues from five selected control and 1000 mg/kg bw/day dose group animals and any animals which did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown above from the remaining control and 1000 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

Since there were indications of treatment-related liver and thyroid changes, examination was subsequently extended to include similarly prepared sections of the liver and thyroids from selected animals in the low and intermediate groups.

Microscopic examination was conducted by the Study Pathologist (Roger Alison at Roger Alison Ltd, Caerfyrddin Fach, Cilcennin, Lampeter, SA48 8RN, United Kingdom).
Other examinations:
Reproductive Indices
Mating Performance and Fertility

The following parameters were calculated from the individual data during the mating period of the parental generation:

i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = (number of animals mated / number of animals paired) x 100
Pregnancy Index (%) = (number of pregnant females / number of animals mated) x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:

i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = (number of females delivering live offspring / number of pregnant females) x 100

Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i. Implantation Losses (%)

Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre–implantation loss (%) = (number of corpora lutea – number of implantation sites / number of corpora lutea) x 100

Post–implantation loss (%) = (number of implantation sites – total number of offspring born / number of implantation sites) x 100


ii. Live Birth and Viability Indices

The following indices were calculated for each litter as follows:
Live Birth Index (%) = (number of offspring alive on Day 1 / number of offspring born) x 100
Viability Index (%) = (number of offspring alive on Day 4 / number of offspring alive on Day 1) x 100

iii. Sex Ratio (% males)

Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:
(Number of male offspring / total number of offspring) x 100
Statistics:
See below
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See results
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
See results
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See results
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See results
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See results
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See results
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality
There were no unscheduled deaths.

Clinical Observations
Animals of either sex from all treatment groups showed incidences of increased salivation. The number of animals and frequency of occasions showed a dose related response to treatment.

Functional Observations
Behavioral Assessments

There were no treatment-related changes in the behavioral parameters at 100, 300 or 1000 mg/kg bw/day.

Functional Performance Tests
There were no treatment related changes in functional performance considered to be related to treatment at 100, 300 or 1000 mg/kg bw/day.

Animals of either sex from all treatment groups showed a statistically significant reduction in hind limb grip strength. The statistically significant difference was confined to one out of the three tests and in the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological significance.

Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 100, 300 or 1000 mg/kg bw/day.


Body Weight
No adverse effects were detected in body weight gain for treated males throughout the treatment period or for treated females during maturation.

Females treated with 1000 or 300 mg/kg bw/day showed a statistically significant increase in body weight gain during the first week of treatment and during lactation. An increase in body weight gain is considered not to represent an adverse effect of treatment.

Females treated with 1000 mg/kg bw/day showed a reduction in body weight gain during the final week of gestation. Recovery in body weight gain was evident thereafter with body weight gains during lactation actually being higher than control females.

Food Consumption
No adverse effects were detected in food consumption for treated males throughout the treatment period or for treated females during maturation, gestation or lactation.

Water Consumption
Animals of either sex treated with 1000 or 300 mg/kg bw/day showed increased water consumption. No such effects were detected in animals of either sex treated with 100 mg/kg bw/day.

Reproductive Performance
Mating
No treatment-related effects were detected in mating performance

Fertility
Three females treated with 1000 mg/kg bw/day were not pregnant. These females did not show any implantation sites following staining of the uteri with a 0.5% ammonium polysulphide solution. No histopathological correlates were evident in the female or male reproductive organs which could have been the cause of the infertility however in view of the other effects detected at 1000 mg/kg bw/day an effect of treatment cannot be excluded.

No such effects were detected in females treated with 300 or 100 mg/kg bw/day.

Gestation Length
There were no differences in gestation lengths. The distribution for treated females was comparable to controls. All animals showed gestation lengths of 22 to 23½ days.

Litter Responses
In total all females from the control, 100, and 300 mg/kg bw/day dose groups and five females from the 1000 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.


Offspring Litter Size, Sex Ratio and Viability
One female treated with 1000 mg/kg bw/day had five corpora lutea and one implantation site but did not give birth to any live offspring. Three further females from this treatment group had a total litter loss by Day 3 post partum. The remaining five females maintained a litter to Day 5 post partum. At 1000 mg/kg bw/day there were no effects detected in the number of corpora lutea. A reduction in the number of implantation sites was however evident and subsequently pre implantation loss was statistically significantly increased. Litter size was slightly reduced on Days 1 and 4 of lactation however statistical significance was not achieved. Of the surviving litters offspring survival and development to Day 5 were unaffected.

No such effects were detected in females treated with 300 or 100 mg/kg bw/day.

There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Offspring Growth and Development
As a consequence of the reduced litter size, females treated with 1000 mg/kg bw/day showed a reduction in litter weight on Days 1 and 4 post partum however statistical significance was not achieved. Offspring body weight gain was also slightly reduced and male offspring body weight on Day 4 of lactation was statistically significantly reduced.

No such effects on litter weight or offspring body weight gains were detected in females treated with 300 or 100 mg/kg bw/day.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, cold, pale, weak, generalised bruising, dark, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity. Offspring in two out of the three total litter losses also had no milk in the stomach. The necropsy findings apparent for offspring on the study were typical for the age observed.

Laboratory Investigations
Hematology
No toxicologically significant effects were detected in the hematological parameters examined.

Males treated with 1000 mg/kg bw/day showed a statistically significant increase in platelet count and statistically significant reductions in hemoglobin and mean corpuscular hemoglobin concentration. Females treated with 1000 mg/kg bw/day showed statistically significant reductions in mean corpuscular hemoglobin and prothrombin time. Females treated with 1000 and 300 mg/kg bw/day also showed a statistically significant increase in platelet count. The majority of the individual values were within normal range for rats of the strain and age used, and in the absence of any associated microscopic changes, the intergroup differences were considered not to be of toxicological importance.

Blood Chemistry
Females treated with 1000 mg/kg bw/day showed an increase in cholesterol and a statistically significant increase in bile acid. Four out of the five individual values for both of these parameters were outside of the normal range for rats of the strain and age used.

No toxicologically significant effects were detected in males treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 100 mg/kg bw/day.

Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in cholesterol and a statistically significant increase in bile acid. Females from this treatment group showed a statistically significant increase in albumin and a statistically significant reduction in chloride concentration. Females treated with 300 mg/kg bw/day also showed a statistically significant increase in bile acid and statistically significant reductions in calcium concentration and phosphorus. The majority of the individual values were within normal ranges for rats of the strain and age used. Although there was evidence of microscopic hepatic changes in treated animals these were considered adaptive therefore the intergroup differences in the blood chemical parameters measured were considered not to be toxicologically significant.

Pathology
Necropsy
Offspring
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Adults
All males and eleven females treated with 1000 mg/kg bw/day, two males and three females treated with 300 mg/kg bw/day and three males and one female treated with 100 mg/kg bw/day had an enlarged liver at necropsy.

One control male, two control females, two females treated with 100 mg/kg bw/day, three females treated with 300 mg/kg bw/day and one female treated with 1000 mg/kg bw/day had reddened lungs at necropsy. The female treated with 1000 mg/kg bw/day and another female from this treatment group also had pale adrenals. A further female treated with 1000 mg/kg bw/day had reddened mandibular lymph nodes and a male from this treatment group had small seminal vesicles. In the absence of any associated histopathological correlates the intergroup differences were considered of no toxicological significance.

Organ Weights
Animals of either sex treated with 1000 and 300 mg/kg bw/day and males treated with 100 mg/kg bw/day showed an increase in liver weight both absolute and relative to terminal body weight. Males treated with 1000 mg/kg bw/day also showed an increase in thyroid weight both absolute and relative to terminal body weight.

No such effects were detected in females treated with 100 mg/kg bw/day.

Males treated with 1000 mg/kg bw/day showed a statistically significant increase in kidney weight both absolute and relative to terminal body weight. Males treated with 1000 and 300 mg/kg bw/day also showed a statistically significant increase in spleen weight both absolute and relative to terminal body weight. Females treated with 1000 mg/kg bw/day showed a statistically significant increase in absolute and relative uterus and cervix weight. In the absence of a true dose related response or any associated histopathological correlates the intergroup differences were considered not to be toxicologically significant.

Histopathology
The following treatment related microscopic findings were observed:

Liver: Hepatocellular hypertrophy, consistent with enzyme induction was evident in animals of either sex from all treatment groups.

Thyroids: Follicular cell hypertrophy/hyperplasia was evident in animals of either sex from all treatment groups. This was considered likely to be secondary to the liver change (due to increased metabolism of thyroid hormones).

Other findings recorded in this study were considered to be within the normal range of background alterations that is seen in untreated animals of this age and strain. In particular, qualitative examination of the stages of spermatogenesis in the testes did not reveal any treatment related abnormalities in the integrity of the various cell types present within the differences stages of the sperm cycle. No treatment related microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea of the ovaries.

No histopathological abnormalities were observed to explain the failure of several high dose animal pairs to breed successfully.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: For reproductive toxicity.
Critical effects observed:
not specified
Discussion

The oral administration of trichloro(N,N-dimethyloctylamine)boron (TK 12146)to rats for a period of up to seven weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels of up to 1000 mg/kg bw/day, resulted in treatment-related microscopic effects in animals of either sex from all treatment groups.

 

Clinical signs were confined to increased salivation detected in animals of either sex from all treatment groups. Water consumption was also increased in animals of either sex treated with 1000 or 300 mg/kg bw/day. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and do not necessarily represent an adverse effect of treatment.

 

No adverse effect was apparent for body weight or food consumption in treated males throughout the treatment period or in treated females during maturation. A reduction in body weight gain was evident however in females treated with 1000 mg/kg bw/day during the final week of gestation. Food consumption was not adversely affected during this period. Parental body weight on Days 0, 7 and 14 of gestation were comparable to control females therefore the reduction in body weight gain during the final week of gestation is most likely to be a result of the slightly reduced litter size at this dosage. 

 

Although there were some statistically significant differences in treated animals from controls for the hematological parameters measured, these differences were considered not to be of toxicological significance. Macroscopic findings detected at necropsy were confined to enlarged livers in a number of animals of either sex from all treatment groups.

 

Histopathological examination of the liver revealed hepatocellular hypertrophy in animals of either sex from all treatment groups. Organ weight data supported this finding with increased absolute and relative liver weights observed in animals of either treated with 1000 and 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day. Females treated with 1000 mg/kg bw/day also showed increases in cholesterol and bile acids with the majority of individual values being outside of the normal range for rats of the strain and age used. In the absence of any degenerative or inflammatory changes, this condition is considered to be adaptive in nature. Microscopic examination of the thyroid revealed follicular cell hypertrophy/ hyperplasia in animals of either sex from all treatment groups. Thyroid weights were also increased in males treated with 1000 mg/kg bw/day. The thyroid and liver changes are characteristic of a consequence of hepatocellular induction as a result of enhanced hepatic metabolism. As a side effect of hepatic induction an increased liver metabolism of thyroid hormones T3 and T4 can occur. This subsequently leads to an enhanced thyroid gland production of these hormones as a consequence of a negative feedback stimulation of TSH production. The appearance of thyroid follicular cell hypertrophy is considered to be a result of this process and was considered to be adaptive in nature. Due to the changes detected in this study, a ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 1000 mg/kg bw/day for systemic toxicity.

 

Mating performance was unaffected by treatment however three females treated with 1000 mg/kg bw/day were non pregnant following positive evidence of mating and one female treated with 1000 mg/kg bw/day had corpora lutea and one implantation site but did not appear to give birth to any live offspring. Three further females from this treatment group had a total litter loss by Day 3post partum. Corpora lutea counts for 1000 mg/kg bw/day females were comparable to controls however pre-implantation loss was increased and subsequently the number of implantation sites were slightly reduced. Litter size at birth, Day 1 and Day 4 of lactation was slightly lower. A true treatment related effect on embryo lethality can therefore not be excluded. Offspring body weight gain and litter weights at Days 1 and 4post partumwere slightly reduced in litters from females treated with 1000 mg/kg bw/day. For litters reared to Day 5 of age there was no obvious adverse effect on survival or development of the offspring, although the number of litters available for assessment was quite low and the improvedpost partumsurvival may have been due to the lower litter size.

 

Within the confines of this screening study it is not possible to establish the exact cause of the reduced number of implantation sites and subsequent reduced litter size. The increased pre-implantation loss may suggest that viable embryos were not formed or did not successfully implant. No such effects were evident at 300 or 100 mg/kg bw/day therefore based on the findings observed at 1000 mg/kg bw/day the NOEL for reproductive toxicity was considered to be 300 mg/kg bw/day.

Conclusions:
The oral administration of trichloro(N,N-dimethyloctylamine)boron (TK 12146) to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, resulted in treatment-related microscopic effects in animals of either sex from all treatment groups. A No Observed Effect Level (NOEL) was therefore not established in either sex. The organ weight and the microscopic liver and thyroid changes detected in treated animals were considered to be an adaptive response to treatment. In terms of risk assessment, these findings observed on this study would suggest that a No Observed Adverse Effect Level (NOAEL) for systemic toxicity can be established at 1000 mg/kg bw/day for animals of either sex because the findings were not evidence of true systemic toxicity.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 300 mg/kg bw/day.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

Methods….

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

 

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. 

 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

 

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

 

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

 

Adult males were terminated on Day 43 or Day 44, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Food Consumption

No adverse effects were detected in food consumption for treated males throughout the treatment period or for treated females during maturation, gestation or lactation.

 

Water Consumption

Animals of either sex treated with 1000 and 300 mg/kg bw/day showed increased water consumption. No such effects were detected in animals of either sex treated with 100 mg/kg bw/day.

 

Reproductive Performance

Mating

No treatment-related effects were detected in mating performance.

 

Fertility

Three females treated with 1000 mg/kg bw/day were not pregnant.

No such effects were detected in females treated with 300 or 100 mg/kg bw/day.

  

Gestation Lengths

Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.

 

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

One female treated with 1000 mg/kg bw/day had corpora lutea and one implantation site but did not give birth to any live offspring. Three further females from this treatment group had a total litter loss by Day 3 post partum. The remaining five females maintained a litter to Day 5 post partum.

 

Females treated with 1000 mg/kg bw/day showed a reduction in the number of implantation sites due to an increase in pre implantation loss. Subsequently litter size was slightly reduced at 1000 mg/kg bw/day at birth and on Days 1 and 4 of lactation.

Viability indices of treated females were however comparable to control females. No differences in sex ratio was evident for offspring from treated litters when compared to those from the controls.

 

Offspring Growth and Development

As a consequence of the reduced litter size, females treated with 1000 mg/kg bw/day showed a slight reduction in litter weight on Days 1 and 4 post partum. Offspring body weight gain at this dose level was also slightly reduced between Days 1 and 4 post partum. No such effects were detected in litters from females treated with 300 or 100 mg/kg bw/day. Surface righting was comparable to controls. The clinical sign and necropsy findings apparent for offspring on the study were typical for the age observed. 

 

Laboratory Investigations

Hematology

No toxicologically significant effects were detected in the hematological parameters examined.

  

Blood Chemistry

Females treated with 1000 mg/kg bw/day showed an increase in cholesterol and bile acids. No toxicologically significant effects were detected in males treated with 1000 mg/kg bw/day or in animals of either sex treated with 300 or 100 mg/kg bw/day.

 

Pathology

Necropsy

At necropsy the most noted finding was enlarged livers in all males and eleven females treated with 1000 mg/kg bw/day, in two males and three females treated with 300 mg/kg bw/day and in three males and one female treated with 100 mg/kg bw/day.

 

Organ Weights

Animals of either sex treated with 1000 and 300 mg/kg bw/day and males treated with 100 mg/kg bw/day showed an increase in liver weight both absolute and relative to terminal body weight.

No such effects were detected in females treated with 100 mg/kg bw/day.

 

Histopathology

The following treatment related microscopic findings were observed: 

 

Liver:Hepatocellular hypertrophy was evident in animals of either sex from all treatment groups.

 

Thyroids:Follicular cell hypertrophy/hyperplasia was evident in animals of either sex from all treatment groups.

 

Conclusion

The oral administration of trichloro(N,N-dimethyloctylamine)boron (TK 12146) to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, resulted in treatment-related microscopic effects in animals of either sex from all treatment groups. A No Observed Effect Level (NOEL) was therefore not established in either sex. The organ weight and the microscopic liver and thyroid changes detected in treated animals were considered to be an adaptive response to treatment. In terms of risk assessment, these findings observed on this study would suggest that a No Observed Adverse Effect Level (NOAEL) for systemic toxicity can be established at 1000 mg/kg bw/day for animals of either sex because the findings were not evidence of true systemic toxicity.

 

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 300 mg/kg bw/day. 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422) with trichloro(N,N-dimethyloctylamine)boron (TK 12146) the NOAEL for systemic toxicity was found to be 1000 mg/kg/day.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only this study is available (OECD 422)

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
In accordance with section 1 of REACh (Regulation (EC) No 1907/2006) Annex XI, the repeated dose toxicity study by inhalation exposure (required in Annex VIII section 8.6.1) does not need to be conducted as oral exposure is the most relevant route.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
In accordance with section 1 of REACh (Regulation (EC) No 1907/2006) Annex XI, the repeated dose toxicity study by inhalation exposure (required in Annex VIII section 8.6.1) does not need to be conducted as oral exposure is the most relevant route.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
In accordance with section 1 of REACh (Regulation (EC) No 1907/2006) Annex XI, the repeated dose toxicity study by dermal exposure (required in Annex VIII section 8.6.1) does not need to be conducted as oral exposure is the most relevant route.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
In accordance with section 1 of REACh (Regulation (EC) No 1907/2006) Annex XI, the repeated dose toxicity study by dermal exposure (required in Annex VIII section 8.6.1) does not need to be conducted as oral exposure is the most relevant route.

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No.1272/2008) and DSD (Directive 67/548/EEC).